Novel sulphur containing lipids for use as food supplement or as medicament

ABSTRACT

The present disclosure relates to lipid compounds of general formula (I) wherein R 1  is chosen from a C 10 -C 22  alkyl group, a C 10 -C 22  alkenyl group having 1-6 double bonds, and a C 10 -C 22  alkynyl group having 1-6 triple bonds; R 2  and R 3  are the same or different and may be selected from different substituents; Y is chosen from sulphur, sulfoxide, and sulfone; and X is a carboxylic acid or a derivative thereof, a carboxylic ester, a carboxylic anhydride, or a carboxamide; or a pharmaceutically acceptable salt, complex, or solvate thereof. The present disclosure also relates to pharmaceutical compositions and lipid compositions comprising such compounds, and methods of using such compounds such as for treating diseases related to cardiovascular, metabolic, and/or inflammatory diseases.

This is a continuation of application Ser. No. 13/054,212, filed Jan.14, 2011, which is a national stage filing under 35 U.S.C. §371 ofInternational Application No. PCT/NO2009/000262, filed on Jul. 13, 2009,which claims the benefit of priority of U.S. Provisional PatentApplication No. 61/080,804 and European Patent Application No.08160450.6, which were filed on Jul. 15, 2008.

TECHNICAL FIELD

The present invention relates to lipid compounds of the general formula(I):

wherein

-   -   R₁ is selected from a C₁₀-C₂₂ alkyl, a C₁₀-C₂₂ alkenyl having        1-6 double bonds, and a C₁₀-C₂₂ alkynyl having 1-6 triple bonds;    -   R₂ and R₃ are the same or different and may be selected from a        group of substituents consisting of a hydrogen atom, a hydroxy        group, an alkyl group, a halogen atom, an alkoxy group, an        acyloxy group, an acyl group, an alkenyl group, an alkynyl        group, an aryl group, an alkylthio group, an alkoxycarbonyl        group, a carboxy group, an alkylsulfinyl group, an alkylsulfonyl        group, an amino group, and an alkylamino group, provided that R₂        and R₃ cannot both be a hydrogen atom; or    -   R₂ and R₃ can be connected in order to form a cycloalkane like        cyclopropane, cyclobutane, cyclopentane or cyclohexane;    -   Y is selected from sulphur, sulfoxide, and sulfone;    -   X represents a carboxylic acid or a derivative thereof, a        carboxylic ester or a carboxamide;        or a pharmaceutically acceptable salt, solvate, solvate of such        salt or a prodrug thereof.

In those cases were R₂ and R₃ are different, the compounds of formula(I) are capable of existing in stereoisomeric forms. It will beunderstood that the invention encompasses all optical isomers of thecompounds of formula (I) and mixtures a thereof.

The invention also relates to pharmaceutical compositions and lipidcompositions comprising such compounds, and to such compounds for use asmedicaments or for use in therapy, in particular for the treatment ofdiseases related to the cardiovascular, metabolic and inflammatorydisease area.

BACKGROUND OF THE INVENTION

Up to date, there has been a lot of research on fatty acid analogues andtheir effects on diverse physiological processes impacting normal healthand chronic diseases.

For example, dietary polyunsaturated fatty acids (PUFAs) have been shownto regulate plasma lipid levels, cardiovascular and immune functions,insulin action, and neuronal development and visual function.

Tetradecylthioacetic acid (TTA) is a modified fatty acid which has anumber of powerful effects demonstrable both in-vivo and in-vitro.

TTA has properties very similar to natural fatty acids, the maindifference being that it cannot be oxidised by the mitochondrialβ-oxidation, but significantly increases the oxidation of other fattyacids. Despite the fact that TTA is not able to undergo β-oxidation, itis metabolised in most ways as a normal saturated fatty acid.

TTA affects oxidative status at different levels by having the potentialof changing the antioxidant defense system, in addition to being anantioxidant itself through its free radical scavenging capacity.

Addition of TTA may prevent the oxidative modification of low-densitylipoprotein (LDL) particles in plasma and reduce the generation of lipidperoxides.

Several polyunsaturated fatty acid derivatives with sulfur in 3-positionhave been prepared (Flock et al, Acta Chemica Scand., 1999, 53, 436).Methyl (all-Z)-3-thia-6,9,12,15-octadecatetraenoate was tested in aWistar rat model, and the effects were compared to the effects of TTA.The results suggest that both the saturated and the unsaturated fattyacids lowered plasma triglycerides to a similar extent (Willumsen et al,J. Lipid Mediators Cell Signalling, 1997, 17, 115)

It has surprisingly been found that novel fatty acid derivativesrepresented by the general formula (I) have higher affinities for thereceptors PPARα and PPARγ compared to TTA and(all-Z)-3-thia-6,9,12,15-octadecatetraenoic acid. Fatty acid derivativesrepresented by the general formula (I) also reduced triglycerid,cholesterol and free fatty acids levels in a dyslipidemic mice model toa greater extent than TTA and(all-Z)-3-thia-6,9,12,15-octadecatetraenoic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Results of PPAR activation in PPARα, PPARδ, and PPARγ luciferasereporter cell lines by compounds according to the present disclosurecompared to PPARα, PPARδ, and PPARγ activity of GW7647, L-165041, andBRL49653, respectively.

FIG. 2: Plasma triglyceride levels and plasma cholesterol levels inAPOE*3Leiden mice after administration of compounds according to thepresent disclosure and unsubstituted reference substances.

FIG. 3: Plasma glucose levels in ob/ob mice after administration of2-((5Z,8Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoic acid,pioglitazone, so and a placebo.

FIG. 4: Plasma insulin levels in ob/ob mice after administration of2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoic acid,pioglitazone, and a placebo.

FIG. 5: Whole blood HbAlc levels in ob/ob mice after administration of2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoic acid,pioglitazone, and a placebo.

FIG. 6: Body weight differences in ob/ob mice after administration of2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoic acid,pioglitazone, and a placebo.

SUMMARY OF THE INVENTION

One object of the present invention is to provide lipid compounds havingimproved biological activity compared to 3-thia fatty acids. This objectis achieved by a lipid compound of formula (I)

In particular, the present invention relates to compounds of formula(I), wherein:

-   -   R₁ is selected from a C₁₀-C₂₂ alkyl, a C₁₀-C₂₂ alkenyl having        1-6 double bonds, and a C₁₀-C₂₂ alkynyl having 1-6 triple bonds;    -   R₂ and R₃ are the same or different and may be selected from a        group of substituents consisting of a hydrogen atom, a hydroxy        group, an alkyl group, a halogen atom, an alkoxy group, an        acyloxy group, an acyl group, an alkenyl group, an alkynyl        group, an aryl group, an alkylthio group, an alkoxycarbonyl        group, a carboxy group, an alkylsulfinyl group, an alkylsulfonyl        group, an amino group, and an alkylamino group, provided that R₂        and R₃ cannot both be a hydrogen atom; or    -   R₂ and R₃ can be connected in order to form a cycloalkane like        cyclopropane, cyclobutane, cyclopentane or cyclohexane;    -   Y is selected from sulphur, sulfoxide, and sulfone;    -   X represents a carboxylic acid or a derivative thereof, a        carboxylic ester or a carboxamide;        or a pharmaceutically acceptable salt, solvate, solvate of such        salt or a prodrug thereof.

In a compound according to the invention, said alkyl group may beselected from the group consisting of methyl, ethyl, n-propyl,isopropyl, n-butyl, sec.-butyl, and n-hexyl; said alkenyl group may beselected from the group consisting of allyl, 2-butenyl, and 3-hexenyl;said alkynyl group may be selected from the group consisting ofpropargyl, 2-butynyl, and 3-hexynyl; said halogen atom may be selectedfrom the group consisting of fluorine, chlorine, bromine, and iodine;said alkoxy group may be selected from the group consisting of methoxy,ethoxy, propoxy, isopropoxy, sec.-butoxy, phenoxy, benzyloxy, OCH₂CF₃,and OCH₂CH₂OCH₃; said acyloxy group may be selected from acetoxy,propionoxy, and butyroxy; said aryl group is a phenyl group; saidalkylthio group may be selected from the group consisting of methylthio,ethylthio, isopropylthio, and phenylthio; said alkoxycarbonyl group maybe selected from the group consisting of methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, and butoxycarbonyl; said alkylsulfinylgroup may be selected from the group consisting of methanesulfinyl,ethanesulfinyl, and isopropanesulfinyl; said alkylsulfonyl group may beselected from the group consisting of methanesulfonyl, ethanesulfonyl,and isopropanesulfonyl; said alkylamino group may be selected from thegroup consisting of methylamino, dimethylamino, ethylamino, anddiethylamino; said carboxylate group may be selected from the groupconsisting of ethyl carboxylate, methyl as carboxylate, n-propylcarboxylate, isopropyl carboxylate, n-butyl carboxylate, sec.-butylcarboxylate, and n-hexyl carboxylate; said carboxamide group may beselected from the group consisting of carboxamide such as N-methylcarboxamide, N,N-dimethyl carboxamide, N-ethyl carboxamide andN,N-diethyl carboxamide.

In one embodiment of the invention, one of the substituents R₂ and R₃ ofthe compound of formula (I) is hydrogen and the other one is selectedfrom a group of substituents consisting of a hydroxy group, an alkylgroup, a halogen atom, an alkoxy group, an acyloxy group, an acyl group,an alkenyl group, an alkynyl group, an aryl group, an alkylthio group,an alkoxycarbonyl group, a carboxy group, an alkylsulfinyl group, analkylsulfonyl group, an amino group, and an alkylamino group.

In a preferred embodiment R₂ and R₃ are independently selected from ahydrogen atom, an alkyl group, an alkoxy group or an aryl group; or R₂and R₃ can be connected in order to form a cycloalkane.

In another preferred embodiment R₂ and R₃ are independently selectedfrom a hydrogen atom, an alkyl group, or a methoxy group or an ethoxygroup.

In yet another preferred embodiment R₂ and R₃ are independently selectedfrom a hydrogen atom, an ethyl, methoxy or ethoxy group, phenyl; or R₂and R₃ are connected to form a cyclobutane group.

In another embodiment of the invention, the substituents R₂ and R₃ ofthe compound of formula (I) are the same or different and may beselected from a group of substituents consisting of a hydroxy group, analkyl group, a halogen atom, an alkoxy group, an acyloxy group, an acylgroup, an alkenyl group, an alkynyl group, an aryl group, an alkylthiogroup, an alkoxycarbonyl group, a carboxy group, an alkylsulfinyl group,an alkylsulfonyl group, an amino group. Preferably R₂ and R₃ are alkylgroups selected from methyl, ethyl, n-propyl, or isopropyl, morepreferably selected from methy or ethyl, and most preferably R₂ and R₃are ethyl.

In one embodiment of the invention the substituent R₁ of the compound offormula (I) is a C₁₀-C₂₂ alkyl, and the said compound is derived from asaturated fatty acid.

Preferably, the substituents R₂ and R₃ of the compound of formula (I)are the same or different and may be selected from a group ofsubstituents as mentioned above, and the substituent R₁ is a C₁₀-C₂₂alkyl, and the said compound is derived from a saturated fatty acid.

When derived from a polyunsaturated fatty acid, R₁ is typically aC₁₀-C₂₂ alkenyl with 2-6 double bonds, e.g. 3-6 double bounds, e.g. 3-6methylene interrupted double bonds in Z configuration. For example, R₁is:

-   -   a C₁₅ alkenyl with 4 methylene interrupted double bonds in        Z-configuration    -   a C₁₈ alkenyl with 3-5 double bonds, e.g. a C₁a alkenyl with 5        methylene interrupted double bonds in Z configuration    -   a C₁₄-C₂₂ alkenyl group with at least one double bond, having Z        configuration, and having the first double bond at the third        carbon-carbon bond from the omega (ω) end of the carbon chain    -   a C₂₀ alkenyl with 5 methylene interrupted double bonds in        Z-configuration    -   a C₂₂ alkenyl with 6 methylene interrupted double bonds in        Z-configuration.

Furthermore, R₁ may be a C₁₀-C₂₂ alkynyl, e.g. a C₁₆-C₂₂ alkynyl with1-6 triple bonds.

In one embodiment of the invention, the substituent Y of the compound offormula (I) is sulfur.

In another embodiment of the invention, the substituent Y of thecompound of formula (I) is sulfoxide.

In still another embodiment of the invention, the substituent Y of thecompound of formula (I) is sulfone.

In one embodiment of the invention, the substituent X of the compound offormula (I) is a carboxylic acid in the form of an ester, a free acid, atriglyceride or a phospholipid.

Preferably, the substituent X is a carboxylic acid in the form of anester, or a free acid, and more preferably X is a carboxylic acid in theform of a free acid.

In another embodiment of the invention, the substituent R₁ is a C₁₀-C₂₂alkyl, and the lipid compound being derived from a saturated fatty acid;R₂ and R₃ are the same or different and may be selected from a group ofsubstituents consisting of a hydroxy group, an alkyl group, a halogenatom, an alkoxy group, an acyloxy group, an acyl group, an alkenylgroup, an alkynyl group, an aryl group, an alkylthio group, analkoxycarbonyl group, a carboxy group, an alkylsulfinyl group, analkylsulfonyl group, an amino group; preferably R₂ and R₃ are alkylgroups; and X is a carboxylic acid in the form of a free acid.

The invention also relates to salts of the compound of formula (I). Suchsalts may be represented by

wherein X is COO⁻,Z⁺ is selected from the group consisting of U⁺, Na⁺, K⁺, NH₄ ⁺,

wherein X═COO⁻, Z²⁺ is selected from the group consisting of Mg²⁺, Ca2⁺,

Another representative salt is

wherein X is COO⁻ Z^(n+) is a polyvalent cation such as

In the case the compounds of formula (I) is in the form of aphospholipid, such compounds may be represented by the followingformulas (II-IV),

wherein Z is:

wherein Z is:

wherein Z is:

Compounds of formula (I), wherein X is a carboxylic acid in the form ofa triglyceride, a 1,2-diglyceride, a 1,3 diglyceride, a 1-monoglycerideand a 2-monoglyceride, are also included in the present invention. Theseare hereinafter represented by the formulas (V), (VI), (VII), (VIII) and(IX), respectively.

The compounds of formula (I) are capable of existing in stereoisomericforms. It will be understood that the invention encompasses all opticalisomers of the compounds of formula (I) and mixtures thereof. Hence,compounds of formula (I) being present as diastereomers, racemates andenantiomers are included.

In a preferred embodiment of the invention the compound of formula (I)is

ethyl 2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoate.

In another preferred embodiment of the invention the compound of formula(I) is

ethyl1-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)-cyclobutanecarboxylate.

The present invention also relates to a lipid compound according offormula (I) for use as a medicament.

In a further aspect, the present invention provides a food supplement, afood additive, or a neutraceutical preparation comprising a lipidcompound of formula (I).

Such a food supplement may be produced for administration through anyroute of administration. For example, the food supplement may beadministered as a liquid nutritional or as a beverage.

The food supplement may be in the form of a capsule, e.g. a gelatinecapsule, and the capsule may be flavoured.

In still a further aspect, the present invention provides apharmaceutical composition comprising a compound of formula (I),preferably together with one or more pharmaceutically acceptablecarriers or excipients.

The novel lipid compounds and compositions of the invention may beformulated in conventional oral administration forms, e.g. tablets,coated tablets, capsules, powders, granulates, solutions, dispersions,suspensions, syrups, emulsions, sprays, etc using conventionalexcipients, e.g. solvents, diluents, binders, sweeteners, aromas, pHmodifiers, viscosity modifiers, antioxidants, corn starch, lactose,glucose, microcrystalline cellulose, magnesium stearate,polyvinylpyrrolidone, citric acid, tartaric acid, water, ethanol,glycerol, sorbitol, polyethylene glycol, propylene glycol, cetylstearylalcohol, carboxymethylcellulose or fatty substances such as hard fat orsuitable mixtures thereof etc. Conventional formulation techniques, wellknown in the art, may be used.

The compositions may likewise be administered by conventionaladministration routes, i.e. orally. The use of orally administrablecompositions, e.g. tablets, coated tablets, capsules, syrups, etc isespecially preferred.

A suitable daily dosage of the compound according to formula (I) is 1 mgto 10 g of said compound; 50 mg to 1 g of said compound, or 50 mg to 200mg of said compound.

The pharmaceutical composition according to the invention may be used asa medicament.

The present invention also relates to lipid composition comprising alipid compound according to formula (I). Suitably, at least 60% byweight, or at least 80% by weight of the lipid composition is comprisedof said compound.

The lipid composition may further comprise a pharmaceutically acceptableantioxidant, e.g. tocopherol.

Further, the present invention relates to a lipid composition for use asa medicament.

Additionally, the present invention relates to the use of a lipidcompound according to formula (I) for use in:

-   -   activation or modulation of at least one of the human peroxisome        proliferator-activated receptor (PPAR) isoforms α, γ or δ,        wherein said compound e.g. is a pan-agonist or modulator    -   the prevention and/or treatment of a dyslipidemic condition,        e.g. hypertriglyceridemia (HTG)    -   the prevention and/or treatment of elevated triglyceride levels,        LDL cholesterol levels, and/or VLDL cholesterol levels    -   the treatment and/or the prevention of obesity or an overweight        condition    -   the reduction of body weight and/or for preventing body weight        gain    -   the treatment and/or the prevention of a fatty liver disease,        e.g. non-alcoholic fatty liver disease (NAFLD).    -   the treatment and/or the prevention of atherosclerosis    -   the prevention of myocardial infarction    -   the treatment and/or the prevention of peripheral insulin        resistance and/or a diabetic condition    -   the treatment and/or prevention of type 2 diabetes    -   the reduction of plasma insulin, blood glucose and/or serum        triglycerides    -   the treatment and/or the prevention of an inflammatory disease        or condition.

The invention also relates to lipid compounds according to formula (I)for the treatment of the above mentioned conditions, and to methods forthe treatment and/or prevention of the conditions listed above,comprising administering to a mammal in need thereof a pharmaceuticallyactive amount of a compound according to formula (I).

In addition, the present invention encompasses methods for manufacturinglipid compounds according to formula (I). The raw material may e.g.originate from a vegetable, a microbial and/or an animal source, such asa marine fish oil. Preferably a marine oil or a krill oil is used.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have found that compounds of formula (I) aspresented above, have remarkably good pharmaceutical activity.

As used herein, the term “lipid compound” relates to fatty acidanalogues derived from e.g. saturated fatty acids, monounsaturated fattyacids, polyunsaturated fatty acids and lipids comprising 1-6 triplebonds.

A “pharmaceutically active amount” relates to an amount that will leadto the desired pharmacological and/or therapeutic effects, i.e. anamount of the combination product which is effective to achieve itsintended purpose. While individual patient needs may vary, determinationof optimal ranges for effective amounts of the combination product iswithin the skill of the art. Generally, the dosage regimen for treatinga condition with the combination product of this invention is selectedin accordance with a variety of factors, including the type, age,weight, sex, diet and medical condition of the patient.

By “a pharmaceutical composition” is meant a lipid compound according tothe invention in any form suitable to be used for a medical purpose.

“Treatment” includes any therapeutic application that can benefit ahuman or non-human mammal. Both human and veterinary treatments arewithin the scope of the present invention. Treatment may be in respectof an existing condition or it may be prophylactic.

Nomenclature and Terminology

Fatty acids are straight chain hydrocarbons possessing a carboxyl (COOH)group at one end (a) and (usually) a methyl group at the other (ω) end.In chemistry, the numbering of the carbon atoms starts from the α end.

The α carbon refers to the first carbon after the carbon that attachesto the functional group, and the second carbon is the β carbon.

As used herein, the expression “methylene interrupted double bonds”relates to the case when a methylene group is located between toseparate double bonds in a carbon chain of a lipid compound.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have surprisingly found that the following lipid compoundshown in categories A-E, are particularly preferable.

Category A

-   -   derived from saturated fatty acids    -   R₁ is a C₁₀-C₂₂ alkyl    -   X represents a carboxylic acid or a derivative thereof, a        carboxylic ester or a carboxamide

Example i

R₁═C₁₄, Y═S

Category B

-   -   derived from monounsaturated fatty acids    -   R₁ is a C₁₀-C₂₂ alkenyl having 1 double bond    -   X represents a carboxylic acid or a derivative thereof, a        carboxylic ester or a carboxamide

Example ii

R₁═C₁₈, Y═S

Example iii

R₁═C₁₄, Y═S

Category C

-   -   derived from polyunsaturated fatty acids    -   R₁ is a C₁₀-C₂₂ alkenyl having 2-6 double bonds    -   X represents a carboxylic acid or a derivative thereof, a        carboxylic ester or a carboxamide

Example iv

R₁═C₂₀ with 5 methylene interrupted double bonds in Z-configuration, Y═S

Example v

R₁═C₂₂ with 6 methylene interrupted double bonds in Z-configuration, Y═S

Example vi

R₁═C₁₅ with 3 methylene interrupted double bonds in Z-configuration, Y═S

Example vii

R═C₁₅ with 4 methylene interrupted double bonds in Z-configuration, Y═S

Example viii

R₁═C₁₅ with 3 methylene interrupted double bonds in Z-configuration and1 double bond in E-configuration, Y═S

Example ix

R₁═C₁₈ with 5 methylene interrupted double bonds in Z-configuration, Y═S

Example x

R₁═C₁₈ with 4 methylene interrupted double bonds in Z-configuration and1 double bond in E-configuration, Y═S

Category D

-   -   derived from lipids containing 1-6 triple bonds    -   R₁ is a C₁₀-C₂₂ alkynyl    -   X represents a carboxylic acid or a derivative thereof, a        carboxylic ester or a carboxamide

Example xi

R₁═C₁₄ with 1 triple bond, Y═S

Category E

-   -   R₁ is selected from a C₁₀-C₂₂ alkyl, a C₁₀-C₂₂ alkenyl having        1-6 double bonds, and a C₁₀-C₂₂ alkynyl having 1-6 triple bonds    -   X represents a carboxylic acid or a derivative thereof, a        carboxylic ester or a carboxamide    -   Y is sulfoxide or sulfone

Example xii

R₁═C₁₅ with 4 methylene interrupted double bonds in Z-configuration,Y═SO

Example xiii

R₁═C₁₅ with 4 methylene interrupted double bonds in Z-configuration,Y═SO₂

Specific examples of preferred lipid compounds according to theinvention are:

Category A—Saturated Fatty Acids:

2-(tetradecylthio)butanoic acid (1)

R₁═C₁₄H₂₉, R₂=ethyl, R₃=a hydrogen atom, Y═S and X═COOH

2-methoxy-2-(tetradecylthio)acetic acid (2)

R₁═C₁₄H₂₉, R₂=methoxy, R₃=a hydrogen atom, Y═S and X═COOH

2-(icosylthio)butanoic acid (3)

R₁═C₂₀H₄₁, R₂=ethyl, R₃=a hydrogen atom, Y═S and X═COOH

2-ethyl-2-(tetradecylthio)butanoic acid (4)

R₁═C₁₄H₂₉, R₂═R₃=ethyl, Y═S and X═COOH

Category B—Monounsaturated Fatty Acids:

2-ethyl-3-thia-12Z-henelcosaenoic acid (5)

R₁═C₁₈H₃₅, R₂=ethyl, R₃=a hydrogen atom, Y═S and X═COOH

(Z)-2-ethyl-2-(octadec-9-enylthio)butanoic acid (6)

R₁═C₁₈H₃₅, R₂═R₃=ethyl, Y═S and X═COOH

Category C—Polyunsaturated Fat Acid Derivatives:

2-((3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenylthio)butanoic acid (7)

R₁═C₁₅H₂₃, R₂=ethyl, R₃=a hydrogen atom, Y═S and X═COOH

2-ethyl-2-((3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenylthio)butanoic acid(8)

R₁═C₁₅H₃₁, R₂═R₃=ethyl, Y═S and X═COOH

2-((5Z,8Z,1Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)propanoic acid(9)

R₁═C₂₀H₃₁, R₂=methyl, R₃=a hydrogen atom, Y═S and X═COOH

2-((5Z,8Z,11Z,4Z,1Z)-icosa-5,8,11,14,17-pentaenylthio)butanoic acid (10)

R₁═C₂₀H₃₁, R₂=ethyl, R₃=a hydrogen atom, Y═S and X═COOH

2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)-2-methylpropanoicacid (11)

R₁═C₂₀H₃₁, R₂=methyl, R₃=methyl, Y═S and X═COOH

2-ethyl-2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoicacid (12)

R₁═C₂₀H₃₁, R₂═R₃=ethyl, Y═S and X═COOH

1-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)cyclobutanecarboxylicacid (13)

R₁═C₂₀H₃₁, R₂ and R₃ combines to form cyclobutane ring, Y═S and X═COOH

2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)-2-phenylaceticacid (14)

R₁═C₂₀H₃₁, R₂=phenyl, R₃=a hydrogen atom, Y═S and X═COOH

2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)-2-methoxyaceticacid (15)

R₁═C₂₀H₃₁, R₂=methoxy, R₃=a hydrogen atom, Y═S and X═COOH

2-((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenylthio)butanoicacid (16)

R₁═C₂₂H₃₃, R₂=ethyl, R₃=a hydrogen atom, Y═S and X═COOH

2-((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenylthio)-2-ethylbutanoicacid (17)

R₁═C₂₂H₃₃, R₂═R₃=ethyl, Y═S and X═COOH

2-((9Z,12Z,15Z)-octadeca-9,12,15-trienylthio)butanoic acid (18)

R₁═C₁₈H₃₁, R₂=ethyl, R₃=a hydrogen atom, Y═S and X═COOH

2-ethyl-2-((9Z,12Z,15Z)-octadeca-9,12,15-trienylthio)butanoic acid (19)

R₁═C₁₈H₃₁, R₂═R₃=ethyl, Y═S and X═COOH

propane-1,2,3-tryltris(2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoate)(20)

R₁═C₂₀H₃₁, R₂=ethyl, R₃=a hydrogen atom, Y═S and X=a carboxylic acid inthe form of a triglyceride

Category D—Triple Bond Containing Fatty Acids:

2-(tetradec-12-ynylthio)butanoic acid (21)

R₁═C₁₄H₂₅, R₂=ethyl, R₃=a hydrogen atom, Y═S and X═COOH

2-ethyl-2-(tetradec-12-ynylthio)butanoic acid (22)

R₁═C₁₄H₂₅, R₂═R₃=ethyl, Y═S and X═COOH

2-methoxy-2-(tetradec-12-ynylthio)acetic acid (23)

R₁═C₁₄H₂₅, R₂=methoxy, R₃=a hydrogen atom, Y═S and X═COOH

Category E—Sulfones and Sulfoxides:

2-((3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenylsulfinyl)butanoic acid(24)

R₁═C₁₅H₂₃, R₂=ethyl, R₃=a hydrogen atom, V═SO and X═COOH

2-((3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenylsulfonyl)butanoic acid(25)

R₁═C₁₅H₂₃, R₂=ethyl, R₃=a hydrogen atom, Y═SO₂ and X═COOH

2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenysulfinyl)butanoic acid(26)

R₁═C₂₀H₃₁, R₂=ethyl, R₃=a hydrogen atom, Y═SO and X═COOH

2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylsulfonyl)butanoicacid (27)

R₁═C₂H₃₁, R₂=ethyl, R₃=a hydrogen atom, Y═SO₂ and X═COOH

2-ethyl-2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylsulfinyl)butanoicacid (28)

R₁═C₂₀H₃₁, R₂═R₃=ethyl, Y═SO and X═COOH

2-ethyl-2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylsulfonyl)butanoicacid (29)

R₁═C₂₀H₃₁, R₂═R₃=ethyl, Y═SO₂ and X═COOH

The compounds of categories A-E above, were R₂ and R₃ are different, arecapable of existing in stereoisomeric forms, i.e. all optical isomers ofthe compounds and mixtures thereof are encompassed. Hence, the saidcompounds may be present as diastereomers, racemates and enantiomers.

General Synthetic Methods for the Compounds Described Herein

The compounds of general formula (I) can be prepared by the followinggeneral procedures:

Method I

Method II

The alcohols described in method I and II may be prepared directly fromthe carboxylic esters of, for example, naturally occurring fatty acids;e.g. alpha-linolenic acid, conjugated linoleic acid, eicosapentaenoicacid (EPA), etc. by reduction with a reducing agent like lithiumaluminiumhydride or diisobultylaluminiumhydride at −10 to 0° C. Thealcohols can also be prepared by degradation of the polyunsaturatedfatty acids EPA and DHA, as described by Holmeide et al. (J. Chem. Soc.,Perkin Trans. 1, 2000, 2271). In this case one can start with purifiedEPA or DHA, but it is also possible to start with fish oil containingEPA and DHA in mixture.

Compounds of formula (X) and (XI) are commercially available, or theyare known in the literature, or they are prepared by standard processesknown in the art. The leaving group (LG) present in compounds of formula(XI) may, for example, be mesylate, tosylate or a suitable halogen, suchas bromine.

Using method I, the resulting alcohols can be converted, usingfunctional group interconversion, by methods familiar to persons skilledin the art (step I), to compounds where the terminal hydroxy group havebeen transformed into a suitable leaving group (LG). Suitable leavinggroups include bromine, mesylate and tosylate. These compounds can bereacted further (step II) in a substitution reaction with theappropriately substituted thiol acetic acid derivatives (X), in thepresence of base.

Using method II, the alcohols can be converted to the correspondingthiols (step IV) by methods familiar to persons skilled in the art. Thethiols can then be reacted further (step V) in a substitution reactionwith compounds of formula (XI), in the presence of base in anappropriate solvent system.

The corresponding sulfoxides and sulfones (Y═SO or SO₂) can be preparedby oxidation of the thioethers (Y═S) with a suitable oxidising agent(step III). Examples of oxidising agents are m-chloro-perbenzoic acid(MCPBA), hydrogen peroxide (H₂O₂) and oxone (potassiumperoxymonosulfate). By using 1 equivalent or less of the oxidisingagent, the main product will be the sulfoxide. By using an excessoxidising agent, the main product will be the sulfone.

If the acid derivatives used are carboxylic esters, hydrolysis can beperformed to obtain the free fatty acids. An esterifying group such as amethyl of an ethyl group may be removed, for example, by alkalinehydrolysis using a base such as an alkali metal hydroxide, for exampleLiOH, NaOH or KOH or by using an organic base, for example Et₃N togetherwith an inorganic salt, for example LiCl in an appropriate solventsystem. A tert-butyl group may be removed, for example, by treatmentwith an acid, for example an organic acid such as trifluoroacetic acidor formic acid in an appropriate solvent system. An arylmethyl groupsuch as a benzyl group may be removed, for example, by hydrogenationover a catalyst such as palladium-on-carbon in an appropriate solventsystem.

The preparation of compounds of formula I, according to method I or II,may result in mixtures of stereoisomers. If required, these isomers maybe separated by means of chiral resolving agents and/or by chiral columnchromatography through methods known to the person skilled in the art.

Method III

The compounds of formula (I) wherein X is a carboxylic acid and in theform of a phospholipid can be prepared through the following processes.

Acylation of sn-glycero-3-phosphocholine (GPC) with an activated fattyacid, such as fatty acid imidazolides, is a standard procedure inphosphatidylcholine synthesis. It is a usually carried out in thepresence of DMSO anion with DMSO as solvent (Hermetter; Chemistry andPhysics of lipids, 1981, 28, 111). Sn-Glycero-3-phosphocholine, ascadmium (II) adduct can also be reacted with the imidazolide activatedfatty acid in the presence of DBU (1,8-diazabicyclo[5.4.0]undec-7-ene)to prepare the phosphatidylcholine of the respective fatty acid(International application number PCT/GB2003/002582). Enzymatictransphosphatidylation can effect the transformation ofphosphatidylcholine to phosphatidyletanolamine (Wang et al, J. Am. Chem.Soc., 1993, 115, 10487).

Phospholipids may also be prepared by enzymatic esterification andtransesterification of phospholipids or enzymatic transphosphatidylationof phospholipids. (Hosokawa, J. Am. Oil Chem. Soc. 1995, 1287,Lilja-Hallberg, Biocatalysis, 1994, 195).

Method IV

The compounds of formula (I) wherein X is a carboxylic acid in the formof a triglyceride can be prepared through the following process. Excessof the fatty acid can be coupled to glycerol using dimethylaminopyridine(DMAP) and2-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU).

Method V

The compounds of formula (I) wherein X is a carboxylic acid in the formof a diglyceride can be prepared by reaction of the fatty acid (2equivalents) with glycerol (1 equivalent) in the presence of1,3-dicyclohexylcarbondiimide (DCC) and 4-dimethylaminopyridine (DMAP).

Method VI

The compounds of formula (I) wherein X is a carboxylic acid and in theform of a monoglyceride can be prepared through the following processes.

Acylation of 1,2-O-isopropylidene-sn-glycerol with a fatty acid usingDCC and DMAP in chloroform gives a monodienoylglycerol. Deprotection ofthe isopropylidene group can be done by treating the protected glycerolwith an acidic (HCl, acetic acid etc.) (O'Brian, J. Org. Chem., 1996,5914).

There are several synthetic methods for the preparation ofmonoglycerides with the fatty acid in 2-position. One method utilizesesterification of the fatty acid with glycidol in the presence of1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (EDC) and4-dimethylaminopyridine (DMAP) to produce a glycidyl derivative.Treatment of the glycidyl derivative with trifluoroacetic anhydride(TFAA) prior to trans-esterification the monoglyceride is obtained(Parkkar et al, Bioorg. Med. Chem. Lett. 2006, 2437).

Further methods for the preparation of mono-, di- and tri-glycerides offatty acid derivatives are described in international patentapplication, PCT/FR02/02831.

It is also possible to use enzymatic processes (lipase reactions) forthe transformation of a fatty acid to a mono-, di-, tri-glyceride. A1,3-regiospecific lipase from the fungus Mucor miehei can be used toproduce triglycerides or diglycerides from polyunsaturated fatty acidsand glycerol. A different lipase, the non-regiospecific yeast lipasefrom Candida antartica is highly efficient in generating triglyceridesfrom polyunsaturated fatty acids (Haraldsson, Pharmazie, 2000, 3).

Preparation, Characterization and Biological Testing of Specific FattyAcid Derivatives of Formula (I)

The invention will now be further described by the followingnon-limiting examples, in which standard techniques known to the skilledchemist and techniques analogous to those described in these examplesmay be used where appropriate. Unless otherwise stated:

-   -   evaporations were carried out by rotary evaporation in vacuo;    -   all reactions were carried out at room temperature, typically in        the range between 18-25° C. with solvents of HPLC grade under        anhydrous conditions;    -   column chromatography were performed by the flash procedure on        silica gel 40-63 μm (Merck) or by an Armen Spotflash using the        pre-packed silica gel columns “MiniVarioFlash”,        “SuperVarioFlash”, “SuperVarioPrep” or “EasyVarioPrep” (Merck);    -   yields are given for illustration only and are not necessarily        the maximum attainable;    -   the nuclear magnetic resonance (NMR) shift values were recorded        on a Bruker Avance DPX 200 or 300 instrument, and the peak        multiplicities are shown as follows: s, singlet; d, doublet; dd,        double doublet; t, triplet; q, quartet; p, pentet; m,        multiplett; br, broad;    -   the mass spectra were recorded with a LC/MS spectrometer.        Separation was performed using a Agilent 1100 series module on a        Eclipse XDB-C18 2.1×150 mm column with gradient elution. As        eluent were used a gradient of 5-95% acetonitrile in buffers        containing 0.01% trifluoroacetic acid or 0.005% to sodium        formate. The mass spectra were recorded with a G 1956 A mass        spectrometer (electrospray, 3000 V) switching positive and        negative ionization mode.

Preparation of Intermediates Example 1 Preparation ofS-(3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenyl ethanethioate

Triphenylphosphine (PPh₃) (41.7 g, 159 mmol) was dissolved in drytetrahydrofurane (THF) (250 mL) at 0° C. under inert atmosphere andadded diisopropyl azodicarboxylate (DIAD) (30.8 mL, 159 mmol). Themixture was stirred at 0° C. for 40 minutes and then dropwise added asolution of (all-Z)-3,6,9,12-pentadecatetraenol (17.5 g, 79.4 mmol) andthioacetic acid (11.4 mL, 159 mmol) in dry THF (150 mL). The resultingturbid mixture was stirred at 0° C. for 40 minutes, then at ambienttemperature for two hours. Heptane was added (300 mL), the mixture wasstirred for ten minutes and the precipitated white solid was removed byfiltration. This procedure was repeated twice and finally the residueafter concentration was stirred in heptane (100 mL) for 16 hours.Filtration and purification of the residue by flash chromatography (1%EtOAc in heptane) provided 13.7 g (62% yield) of the title compound asan oil.

¹H-NMR (200 MHz, CDCl₃): δ 0.96 (t, 3H), 2.05 (m, 2H), 2.31 (s+m, 5H),2.76-2.92 (m, 8H), 5.32-5.45 (m, 8H).

Example 2 Preparation of(3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraene-1-thiol

LiAlH₄ (2.05 g, 54.1 mmol) was suspended in dry diethyl ether (100 mL)at 0° C. under inert atmosphere. To this suspension was added dropwise asolution of S-(3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenyl ethanethioate(13.7 g, 49.2 mmol) in dry diethyl ether (50 mL) and the resulting greymixture was stirred at 0° C. for ten minutes and then at ambienttemperature for 30 minutes. The mixture was cooled to ˜5° C., added 1MHCl until pH=2 and filtrated through a short pad of celite. The pad waswashed with water and diethyl ether, the phases were separated and theaqueous phase was extracted twice with diethyl ether (100 mL each). Thecombined organic extracts were dried (Na₂SO₄), filtered and concentratedunder reduced pressure to afford 7.8 g (67% yield) of the title compoundas oil.

¹H-NMR (200 MHz, CDCl₃): δ 0.96 (t, 3H), 2.06 (m, 2H), 2.39 (m, 2H),2.51 (m, 2H), 2.81 (m, 6H), 5.28-5.54 (m, 8H); MS (ESI): 235 [M−H]⁻.

Example 3 Preparation ofS-(5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenyl ethanethioate

Triphenylphosphine (21.0 g, 80 mmol) was dissolved in dry THF (170 mL)at 0° C. under inert atmosphere and added DIAD (15.8 mL, 80 mmol)dropwise. After 40 minutes at 0° C. the white suspension was addeddropwise to a solution of(5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaen-1-ol (11.5 g, 40 mmol)and thioacetic acid (5.7 mL, 80 mmol) in dry THF (50 mL) during 15minutes. The resulting turbid mixture was stirred at 0° C. for 30minutes, followed by ambient temperature for 1.5 hour. Heptane was added(200 mL), the mixture was stirred for ten minutes and the precipitatedwhite solid removed by filtration and rinsed with heptane (150 mL). Theresidue was concentrated to remove most of the THF and stirred atambient for 18 hours. The mixture was filtered, concentrated and addedheptane (200 mL). The resulting mixture was stirred for 2 hours,filtered and evaporated. The residue was purified by flashchromatography on silica gel, using EtOAc:Heptane (2:98), followed byEtOAc:Heptane (4:96) and finally EtOAc: Heptane (5:95). Concentration ofthe appropriate fractions provided 11.0 g (79% yield) of the titlecompound as oil.

¹H-NMR (300 MHz, CDCl₃): δ 0.95 (t, 3H, J=7.5 Hz), 1.40 (m, 2H), 1.58(m, 2H), 2.06 (m, 4H), 2.29 (s, 3H), 2.77-2.87 (m, 10H), 5.25-5.42 (m,10H); MS (Cl (CH₄)): 387 [M+C₃H₅]⁺, 375 [M+C₂H₅]⁺, 347 [M+H]⁺, 333[M−CH₂]⁺, 305 [R−SH]⁺.

Example 4 Preparation of(5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaene-1-thiol

S-(5Z,8Z,11Z,14Z,17Z)-Icosa-5,8,11,14,17-pentaenyl ethanethioate (7.00g, 20.2 mmol) was dissolved in MeOH (100 mL) by stirring 10 minutesuntil the droplets of oil dissolved, before anhydrous potassiumcarbonate, K₂CO₃ (2.79 g, 20.2 mmol) was added in one portion. Themixture was stirred for 1 hour and 20 minutes at ambient temperature andquenched by addition of 1M HCl (50 mL) and water (150 mL). The whitecloudy mixture was added Et₂O (250 mL) and the phases were separated.The water phase was extracted with Et₂O (2×250 mL). The combined organicphases were washed with brine (250 mL) and dried (MgSO₄). Filtration andevaporation gave the title compound as oil (5.99 g, 97% yield), whichwas used without further purification.

¹H-NMR (300 MHz, CDCl₃): δ 0.96 (t, 3H, J=7.5 Hz), 1.31 (t, 1H, J=7.8Hz), 1.44 (m, 2H), 1.61 (m, 2H), 2.06 (m, 4H), 2.51 (m, 2H), 2.77-2.85(m, 8H), 5.28-5.41 (m, 10H); MS (Cl (CH₄)): 345 [M+C₃H₅]⁺, 333[M+C₂H₅]⁺, 305 [M+H]⁺, 271 [M−SH]⁺.

Example 5 Preparation of(5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenyl methanesulfonate

Et₃N (1.50 mL, 10.8 mmol) and methanesulfonyl chloride (402 μL, 5.20mmol) was added to a solution of(5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaen-1-ol (1.15 g, 4.0 mmol)in CH₂Cl₂ (40 mL) held at 0° C. under nitrogen. The mixture was stirredat 0° C. for one hour, and poured into ice-water (100 g) and the waterphase extracted with Et₂O (50 mL). The combined organic extracts wereadded 0.5M H₂SO₄ (35 mL), the organic phase washed with NaHCO₃ (sat.aq.) (25 mL), before dried (Mg₂SO₄, 10 gram). Filtration andconcentration in vacuo afforded 1.24 gram of crude oil. Purification onArmen, SVP D26 column packed with 30 gram of 15-40 μm Merck silica, flow20 mL/min, UV 210 nm and collecting 15 mL fraction, was performed usinggradient elution: (starting heptane:EtOAc (100:0) and increasing during10 min. to 10% EtOAc, then increasing 5 min. to 20% EtOAc (hold 10min.), then increasing in 5 min. to 40% EtOAc (hold 0 min.). Fractions6-14 afforded 1.16 g (79% yield) of the title compound as oil.

¹H-NMR (300 MHz, CDCl₃): δ 0.97 (t, 3H), 1.50 (m, 2H), 1.75 (m, 2H),2.03-2.15 (m, 4H), 2.76-2.86 (m, 8H), 2.99 (s, 3H), 4.22 (t, 2H),5.27-5.40 (m, 10H); MS (electrospray): 389.2 [M+Na]⁺.

Example 6 Preparation of(4S,5R)-3-((S)-2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoyl)-4-methyl-5-phenyloxazolidin-2-oneand(4S,5R)-3-((R)-2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoyl)-4-methyl-5-phenyloxazolidin-2-one

A mixture of2-((5Z,8Z,11Z,117Z)-icosa-5,8,11,14,17-pentaenylthio)butanoic acid (3.0g, 7.9 mmol) in dry dichloromethane (40 mL) held at 0° C. under nitrogenwas added DMAP (1.0 g, 9.5 mmol) and 1,3-dicyclohexylcarbodiimide (DCC)(1.8 g, 8.7 mmol). The resulting mixture was stirred at 0° C. for 20minutes, (4S,5R)-4-methyl-5-phenyl-2-oxazolidinone (1.7 g, 9.5 mmol) wasadded and the resulting turbid mixture was stirred at ambienttemperature for 24 hours. The mixture was filtrated and concentratedunder reduced pressure to give a crude product containing the desiredproduct as a mixture of two diastereomers. The residue was purified byflash chromatography on Armen Spotflash instrument on silica gel using2% ethyl acetate in heptane as eluent. The two diastereomers wereseparated and the appropriate fractions were concentrated.(4S,5R)-3-((R)-2-((5Z,8Z,11Z,14Z,17Z)-Icosa-5,8,11,14,17-pentaenylthio)butanoyl)-4-methyl-5-phenyloxazolidin-2-oneeluted first and was obtained in 0.95 g (47% yield) as an oil. 1.47 g(67% yield) of(4S,5R)-3-((S)-2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoyl)-4-methyl-5-phenyloxazolidin-2-onewas obtained as an oil.

(4S,5R)-3-((R)-2-((5Z,8Z,11Z,14Z,17Z)-Icosa-5,8,11,14,17-pentaenylthio)butanoyl)-4-methyl-5-phenyloxazolidin-2-one(E1)

¹H-NMR (300 MHz, CDCl₃): δ 0.93-1.06 (m, 9H), 1.45-1.60 (m, 4H),1.75-1.85 (m, 1H), 2.05-2.15 (m, 5H), 2.55-2.70 (m, 2H), 2.87 (m, 8H),4.69 (t, 1H), 4.79 (p, 1H), 5.30-5.45 (m, 10H), 5.72 (d, 1H), 7.32 (m,2H), 7.43 (m, 3H).

(4S,5R)-3-((S)-2-((5Z,8Z,11Z,14Z,17Z)-Icosa-5,8,11,14,17-pentaenylthio)butanoyl)-4-methyl-5-phenyloxazolidin-2-one

¹H-NMR (300 MHz, CDCl₃): δ 0.93 (d, 3H), 0.99 (t, 3H), 1.05 (t, 3H),1.40-1.56 (m, 4H), 1.50-1.75 (m, 1H), 2.00-2.15 (m, 5H), 2.47-2.65 (m,2H), 2.83 (m, 8H), 4.62 (t, 1H), 4.85 (p, 1H), 5.25-5.45 (m, 10H), 5.70(d, 1H), 7.32 (m, 2H), 7.43 (m, 3H).

Preparation of Target Molecules Example 7 Preparation of ethyl2-((3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenylthio)butanoate (30)

A solution of 3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraene-1-thiol (9.80 g,41.5 mmol) in dry dimethylformamide (DMF) (70 mL) at 0° C. under inertatmosphere was added NaH (60% in mineral oil, 1.82 g, 45.6 mmol) andstirred at this temperature for ten minutes. Ethyl bromobutyrate (6.39mL, 43.5 mmol) was added and the mixture was stirred at ambienttemperature for 30 minutes. The mixture was partitioned betweensaturated NH₄Cl (150 mL) and heptane (150 mL). The aqueous layer wasextracted twice with heptane (100 mL each) and the combined organicextract were washed with water (100 mL) and brine (100 mL). The organiclayer was dried (Na₂SO₄), filtrated and concentrated. The residue waspurification by flash chromatography on silica gel (heptane:EtOAc 99:1then 95:5). Concentration of the appropriate fractions afforded 14.1 g(97% yield) of the title compound as oil.

¹H-NMR (200 MHz, CDCl₃): δ 0.92-1.01 (2×t, 6H), 1.27 (t, 3H), 1.60-1.80(m, 1H), 1.80-1.95 (m, 1H), 2.00-2.15 (m, 2H) 2.25-2.45 (m, 2H),2.60-2.75 (m, 2H), 2.80 (m, 6H), 3.15 (t, 1H), 4.17 (q, 2H), 5.31-5.43(m, 8H); MS (ESI): 373 [M+Na]⁺.

Example 8 Preparation of ethyl2-((3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenylsulfonyl)butanoate (31)

Ethyl 2-((3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenylthio)butanoate (2.7g, 7.7 mmol) was dissolved in dry CHCl₃ (40 mL) and the solution wascooled down to −20° C. under inert atmosphere. meta-Chloroperoxybenzoicacid (mCPBA) (˜77%, 4.0 g, 18 mmol) dissolved in dry CHCl₃ (10 mL) wasadded dropwise and the resulting solution was stirred at −20° C. for 30minutes, allowed to slowly reach ambient temperature and then stirredover night. The solvents were evaporated in vacuo, the residue was addedheptane (30 mL) and the resulting white precipitate was removed byfiltration. The filtrate was concentrated in vacuo and the residue wasadded heptane (10 mL). The resulting white precipitate was again removedby filtration. The filtrate was concentrated in vacuo and the residuewas purified by flash chromatography on silica gel (heptane:EtOAc 4:1).Concentration of the appropriate fractions afforded 0.37 g (13% yield)of the title compound as an oil.

¹H NMR (300 MHz, CDCl₃): δ 0.96 (t, 3H), 1.03 (t, 3H), 1.31 (t, 3H),2.02-2.15 (m, 4H), 2.62 (m, 2H), 2.82 (m, 6H), 3.05 (m, 1H), 3.20 (m,1H), 3.70 (dd, J=10.3 Hz, J=4.7 Hz, 1H), 4.28 (q, 2H), 5.26-5.41 (m,7H), 5.46-5.52 (m, 1H); MS (electrospray): 405.2 [M+Na]⁺

Example 9 Preparation of2-((3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenylthio)butanoic acid (7)

Ethyl 2-((3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenylthio)butanoate (14.1g, 40.2 mmol) was dissolved in ethanol (200 mL) and added a solution ofUOH×H₂O (13.5 g, 322 mmol) in water (50 mL). The resulting turbidsolution was stirred at 70° C. under inert atmosphere for 90 minutes,cooled, added water (100 mL) and 3M HCl until pH=2. The mixture wasextracted three times with heptane (100 mL each). The combined organicextracts were dried (Na₂SO₄), filtered and concentrated under reducedpressure to afford 11.8 g (91% yield) of the title compound as oil.

¹H-NMR (200 MHz, CDCl₃): δ 0.91-1.06 (2×t, J=7.2 Hz, J=7.5 Hz, 6H),1.60-1.80 (m, 1H), 1.80-1.95 (m, 1H), 2.05 (p, J=7.2 Hz, 2H), 2.35 (m,2H), 2.60-2.75 (m, 2H), 2.75-2.90 (m, 6H), 3.14 (t, J=7.1 Hz, 1H),5.31-5.47 (m, 8H); MS (ESI): 321 [M−H]⁻.

Example 10 Preparation of2-((3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenylsulfinyl)butanoic acid(24)

2-((3Z,6Z,9Z,12Z)-Pentadeca-3,6,9,12-tetraenylthio)butanoic acid (0.20g, 0.62 mmol) was dissolved in dry CHCl₃ (10 mL) and the solution wascooled down to −20° C. under inert atmosphere. mCPBA (˜77%, 0.15 g, 0.68mmol) dissolved in dry CHCl₃ (2 mL) was added dropwise and the resultingsolution was stirred at −20° C. for 35 minutes. The solvents wereevaporated in vacuo, the residue was added heptane (10 mL) and theresulting white precipitate was removed by filtration. The filtrate wasconcentrated in vacuo and the residue was added heptane (10 mL). Theresulting white precipitate was again removed by filtration. Thefiltrate was concentrated in vacuo and the residue was purified by flashchromatography on silica gel (heptane:EtOAc+/1% HCOOH 4:1-1:1).Concentration of the appropriate fractions afforded 100 mg (48% yield)of the title compound as an oil.

¹H NMR (200 MHz, CDCl₃): δ 0.95 (t, 3H), 1.10 (2×q, 3H), 1.70-1.80 (m,1H), 2.05 (m, 3.5H), 2.20-2.40 (m, 0.5H), 2.60 (m, 2H), 2.81 (m, 7H),2.90-3.00 (m, 0.5H), 3.10-3.25 (m, 1H), 3.70 (dd, 0.5H), 5.25-5.55 (m,8H);O MS (electrospray): 337.1 [M−H]⁻

Example 11 Preparation of2-((3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenylsulfonyl)butanoic acid(25)

Ethyl 2-((3Z,6Z,9Z,12Z)-pentadeca-3,6,9,12-tetraenylsulfonyl)butanoate(370 mg, 0.97 mmol) was dissolved in ethanol (10 mL) and added asolution of LiOH in H₂O (1M, 3.9 mL, 3.9 mmol). The resulting mixturewas stirred at 60° C. for three hours, cooled, added 0.1M HCl until pH=2and extracted twice with diethyl ether (15 mL each). The combinedorganic layer was washed with brine (15 mL), dried, filtrated,concentrated in vacuo and purified by flash chromatography on silica gel(heptane:EtOAc/5% HCOOH 4:1). Concentration of the appropriate fractionsafforded 250 mg (73% yield) of the title compound as an oil.

¹H NMR (300 MHz, CDCl₃): δ 0.96 (t, 3H), 1.09 (t, 3H), 2.02-2.25 (m,4H), 2.65 (m, 2H), 2.82 (m, 6H), 3.10 (m, 1H), 3.20 (m, 1H),

Example 12 Preparation of ethyl2-((5Z,8Z,1Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)propanoate (32)

(5Z,8Z,11Z,14Z,17Z)-Icosa-5,8,11,14,17-pentaene-1-thiol (305 mg, 1.00mmol) was added to a solution of NaH (60% in mineral oil, 44 mg, 1.10mmol) in dry DMF (10 mL) held at 0° C. under inert atmosphere. After tenminutes ethyl bromopropionate (136 μL, 1.05 mmol) was added and themixture was stirred for 1.5 hour at 0° C. The reaction mixture was addedsat. aq. NH₄Cl (20 mL) and heptane (50 mL). The phases were separatedand the water phase extracted with heptane (2×25 mL). The combinedorganics were washed with brine (25 mL), dried (MgSO₄), filtered andevaporated to give 376 mg of title compound as crude oil. Purificationby flash chromatography on silica gel using gradient elution (startingpure heptane and increasing stepwise to heptane:EtOAc 95:5) afforded 318mg (79% yield) of the title compound as oil.

¹H-NMR (300 MHz, CDCl₃): δ 0.95 (t, 3H), 1.25 (t, 3H), 1.41 (d, 3H),1.44 (m, 2H), 1.58 (m, 2H), 2.06 (m, 4H), 2.60 (m, 2H), 2.71-2.85 (m,8H), 3.36 (d, 1H), 4.17 (m, 2H), 5.25-5.40 (m, 10H); MS (Cl (CH₄)): 445[M+C₃H₅]⁺, 433 [M+C₂H₅]⁺, 405 [M+H]⁺, 359 [M−OEt]⁺, 331 [M−CO₂Et]⁺, 303[R—S]^(+).

Example 13 Preparation of ethyl2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoate (33)

To a solution of (5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaene-1-thiol(305 mg, 1.00 mmol) in dry DMF (10 mL) at 0° C. under inert atmospherewas added NaH (60% In mineral oil, 44 mg, 1.1 mmol). After fifteenminutes ethyl bromobutyrate (154 μL, 1.05 mmol) was added. The mixturewas stirred for 1 hour at 0° C. Sat. aq. NH₄Cl (20 mL), water (20 mL)and heptane (50 mL) were added. The phases were separated and the waterphase was extracted with heptane (2×25 mL). The combined organics werewashed with water (25 mL) and brine (25 mL), dried (MgSO₄), filtered andevaporated to give 379 mg of the title compound as a crude oil.Purification by flash chromatography on silica gel using gradientelution (starting pure heptane and increasing stepwise to heptane:EtOAc95:5) afforded 345 mg (82% yield) of the title compound as oil.

¹H-NMR (300 MHz, CDCl₃): δ 0.93-1.00 (m, 6H), 1.25 (t, 3H), 1.44 (m,2H), 1.59 (m, 2H), 1.68 (m, 1H), 1.87 (m, 1H), 2.07 (m, 4H), 2.57 (m,2H), 2.73-2.88 (m, 8H), 3.12 (m, 1H), 4.17 (m, 2H), 5.27-5.46 (m, 10H);MS (Cl (CH₄)): 459 [M+C₃H₅]⁺, 447 [M+C₂H₅]⁺, 419 [M+H]⁺, 373 [M−OEt]⁺,345 [M−CO₂Et]⁺, 303 [R−S]^(+).

Example 14 Preparation of2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoic acid(10)

Ethyl 2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoate(209 mg, 0.50 mmol) was dissolved in ethanol (2.5 mL) and added to asolution of UOH×H₂O (168 mg, 4.0 mmol) in water (2.5 mL). The resultingturbid solution was stirred at 70° C. under inert atmosphere for 2hours, cooled and added water (10 mL) and 1M HCl (5 mL) to pH=1-2. Themixture was extracted with heptane (2×20 mL) and diethyl ether (20 mL).The combined organic extracts were dried (MgSO₄), filtered andconcentrated under reduced pressure to give 154 mg of the title compoundas crude oil. Purification by flash chromatography on silica gel usinggradient elution (starting with pure heptane and increasing stepwise toheptane:EtOAc (with 5% HOAc) 80:20) afforded 151 mg (77% yield) of thetitle compound as oil.

¹H-NMR (300 MHz, CDCl₃): δ 0.95 (t, 3H), 1.02 (t, 3H), 1.46 (m, 2H),1.52-1.78 (m, 3H), 1.90 (m, 1H), 2.05 (m, 4H), 2.63 (m, 2H), 2.75-2.90(m, 8H), 3.14 (t, 1H) (m, 1H), 4.17 (m, 2H), 5.27-5.46 (m, 10H).

Example 15 Preparation of(S)-2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoicacid (34)

Hydrogen peroxide (30% in water, 0.71 mL, 6.91 mmol) and lithiumhydroxide monohydrate (0.15 g, 3.46 mmol) was added to a solution of(4S,5R)-3-((S)-2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoyl)-4-methyl-5-phenyloxazolidin-2-one(0.95 g, 1.73 mmol) in tetrahydrofuran (12 mL) and water (4 mL) held at0° C. under nitrogen. The reaction mixture was stirred at 0° C. for 30minutes. 10% Na₂SO₃ ((30 mL) was added, the pH was adjusted to ˜2 with5M HCl and the mixture was extracted twice with heptane (30 mL). Thecombined organic extract was dried (Na₂SO₄), filtered and concentrated.The residue was subjected to flash chromatography on silica gel usingincreasingly polar mixtures of heptane and ethyl acetate (98:8→1:1) aseluent. Concentration of the appropriate fractions afforded 0.15 g (17%yield) of the title product as an oil.

¹H-NMR (300 MHz, CDCl₃): δ1.00 (t, 3H), 1.07 (t, 3H), 1.46 (m, 2H),1.60-1.75 (m, 3H), 1.85 (m, 1H), 2.10 (m, 4H), 2.66 (m, 2H), 2.80-2.90(m, 8H), 3.21 (t, 1H), 5.35-5.45 (m, 10H); MS (electrospray): 389.3[M−H]⁻; [α]_(D)-49° (c=0.12, ethanol).

Example 16 Preparation of(R)-2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoicacid (35)

Hydrogen peroxide (30% in water, 1.04 mL, 10.2 mmol) and lithiumhydroxide monohydrate (0.21 g, 5.09 mmol) was added to a solution of(4S,5R)-3-((R)-2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoyl)-4-methyl-5-phenyloxazolidin-2-one(1.40 g, 2.55 mmol) in tetrahydrofuran (15 mL) and water (5 mL) held at0° C. under nitrogen. The reaction mixture was stirred at 0° C. for 45minutes. 10% Na₂SO₃ (a (35 mL) was added, pH was adjusted to ˜2 with 5MHCl and the mixture was extracted twice with heptane (35 mL). Thecombined organic extract was dried (Na₂SO₄), filtered and concentrated.The residue was subjected to flash chromatography on silica gel usingincreasingly polar mixtures of heptane and ethyl acetate (98:84-1:1) aseluent. Concentration of the appropriate fractions afforded 0.17 g (22%yield) of the title product as an oil. ¹H-NMR (300 MHz, CDCl₃): δ 1.00(t, 3H), 1.07 (t, 3H), 1.46 (m, 2H), 1.60-1.75 (m, 3H), 1.85 (m, 1H),2.10 (m, 4H), 2.66 (m, 2H), 2.80-2.90 (m, 8H), 3.21 (t, 1H), 5.35-5.45(m, 10H); MS (electrospray): 389.3 [M−H]⁻; [α]_(D)+50° (c=0.14,ethanol).

Example 17 Preparation of ethyl2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)-2-methylpropanoate(36)

To a solution of (5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaene-1-thiol(305 mg, 1.00 mmol) in dry DMF (10 mL) at 0° C. under inert atmospherewas added NaH (60% in mineral oil, 44 mg, 1.1 mmol). After fifteenminutes ethyl 2-bromo-2-methylbutyrate (154 μL, 1.05 mmol) was added andthe mixture was stirred for 1.5 hour at 0° C. The reaction mixture wasquenched by addition of sat. aq. NH₄Cl (20 mL). Water (20 mL) andheptane (50 mL) were added and the phases were separated. The waterphase was extracted with heptane (2×25 mL). The combined organics werewashed with water (25 mL) and brine (2×25 mL), dried (MgSO₄), filteredand evaporated to give 377 mg of the title compound as a crude oil.Purification by flash chromatography on silica gel using isocraticelution (heptane:EtOAc 98:2) afforded 307 mg (77% yield) of the titlecompound as oil.

¹H-NMR (300 MHz, CDCl₃): δ 0.95 (t, 3H), 1.28 (t, 3H), 1.42 (m, 2H),1.48 (s, 6H), 1.54 (m, 2H), 2.06 (m, 4H), 2.58 (m, 2H), 2.71-2.85 (m,8H), 4.15 (m, 2H), 5.22-5.48 (m, 10H); MS (Cl (CH₄)): 459 [M+C₃H₅]⁺, 447[M+C₂H₅]⁺, 419 [M+H]⁺, 373 [M−OEt]⁺, 345 [M−CO₂Et]⁺, 303 [R−S]^(+).

Example 18 Preparation of2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylhio)-2-methylpropanoicacid (11)

Ethyl2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)-2-methylpropanoate(209 mg, 0.50 mmol) was dissolved in ethanol (2.5 mL) and added to asolution of LiOH×H₂O (168 mg, 4.0 mmol) in water (2.5 mL). The resultingturbid solution was stirred at 70° C. under inert atmosphere for 2hours, cooled and added water (10 mL) and 1M HCl (5 mL) to pH=1-2. Themixture was extracted three times with heptane (3×20 mL). The combinedorganic extracts were dried (MgSO₄), filtered and concentrated underreduced pressure to give 101 mg of the title compound as crude oil.Purification by flash chromatography on silica gel using gradientelution (starting with pure heptane and increasing stepwise toheptane:EtOAc (with 5% HOAc) 80:20) afforded 78 mg (40%) of the titlecompound as oil.

¹H-NMR (300 MHz, CDCl₃): δ 0.95 (t, 3H), 1.35-1.66 (m, 4H), 1.50 (s,6H), 2.07 (m, 4H), 2.63 (t, 3H), 2.70-2.92 (m, 8H), 5.13-5.50 (m, 10H).

Example 19 Preparation of ethyl1-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)cyclobutanecarboxylate(37)

To a solution of (5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaene-1-thiol(305 mg, 1.00 mmol) in dry DMF (10 mL) at 0° C. under inert atmospherewas added NaH (60% in mineral oil, 44 mg, 1.1 mmol). After fifteenminutes ethyl 2-bromo-cyclobutane carboxylate (170 μL, 1.05 mmol) wasadded and the mixture was stirred for 1.5 hour at 0° C. The reaction wasquenched by addition of sat. aq. NH₄Cl (20 mL). Heptane (50 mL) wasadded, and the phases were separated. The water phase was extracted withheptane (2×25 mL). The combined organics were washed with water (25 mL)and brine (25 mL), dried (MgSO₄), filtered and evaporated to give 409 mgof the title compound as a crude oil. Purification by flashchromatography on silica gel using isocratic elution (heptane:acetone98:2) afforded 243 mg (56% yield) of the title compound as oil.

¹H-NMR (300 MHz, CDCl₃): δ 0.95 (t, 3H), 1.27 (t, 3H), 1.42 (d, 3H),1.54 (m, 2H), 1.84 (m, 1H), 1.96-2.23 (m, 7H), 2.51 (m, 2H), 2.60 (m,2H), 2.73-2.90 (m, 8H), 4.18 (m, 2H), 5.23-5.43 (m, 10H); MS (Cl (CH₄)):471 [M+C₂H₅]⁺, 459 [M+C₂H₅]⁺, 431 [M+H]⁺, 385 [M−OEt]⁺, 357 [M−CO₂Et]⁺,303 [R−S]^(+).

Example 20 Preparation of2-ethyl-2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoicacid (12)

NaOEt (21 wt. % in EtOH, 0.37 mL, 0.98 mmol) was added dropwise to asolution of 2-mercapto-2-ethyl butyric acid (0.08 g, 0.49 mmol) in dryEtOH (7 mL) held at 0° C. under inert atmosphere. The resulting mixturewas stirred at 0° C. for 30 minutes before a solution of(5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenyl methanesulfonate (0.15g, 0.41 mmol) in dry EtOH (3 mL) was added dropwise. The resultingturbid mixture was stirred at ambient temperature for 24 hours, pouredinto NH4Cl (sat.) (aq.) (15 mL), added 3M HCl to pH ˜2 before extractedtwice with EtOAc (2×20 mL). The combined organic extracts were washedwith brine (10 mL), dried (MgSO4), filtrated and evaporated in vacuo.The residue was purified by flash chromatography on silica gel using agradient of 10-25% ethyl acetate in heptane as eluent. Concentration ofthe appropriate fractions afforded 0.12 g (70% yield) of the titlecompound as oil.

¹H-NMR (300 MHz, CDCl₃): δ 0.88-1.02 (m, 9H), 1.45-1.58 (2×m, 4H), 1.72(m, 2H), 1.82 (m, 2H) 2.09 (m, 4H), 2.53 (t, 2H), 2.76-2.86 (m, 8H),5.29-5.39 (m, 10H. MS (electrospray): 417.3 [M−H]−;

Example 21 Preparation of ethyl ethyl2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)-2-phenylacetate(38)

To a solution of (5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaene-1-thiol(305 mg, 1.00 mmol) in dry DMF (10 mL) at 0° C. under inert atmospherewas added NaH (60% in mineral oil, 44 mg, 1.1 mmol). After fifteenminutes ethyl 2-bromo-2-phenyl acetate (255 mg, 1.05 mmol) was added andthe mixture stirred for 1.5 hour at 0° C. The reaction mixture wasquenched by addition of sat. aq. NH₄Cl (25 mL). Heptane (50 mL) wasadded and the phases were separated. The water phase was extracted withheptane (2×25 mL). The combined organics were washed with water (25 mL)and brine (25 mL), dried (MgSO₄), filtered and evaporated to give 453 mgof title compound as crude oil. Purification by flash chromatography onsilica gel using isocratic elution (heptane:EtOAc 98:2) afforded 177 mg(38% yield) of the title compound as oil.

¹H-NMR (300 MHz, CDCl₃): δ 0.95 (t, 3H), 1.24 (t, 3H), 1.41 (m, 2H),1.56 (m, 2H), 2.05 (m, 2H), 2.51 (m, 2H), 2.60 (m, 2H), 2.67-2.92 (m,8H), 4.17 (m, 2H), 4.53 (s, 1H), 5.21-5.46 (m, 10H), 7.27-7.35 (m, 3H),7.43-7.46 (m, 2H); MS (Cl (CH₄)): 507 [M+C₃H₅]⁺, 495 [M+C₂H₅]⁺, 467[M+H]⁺, 421 [M−OEt]⁺, 393 [M−CO₂Et]⁺, 303 [R−S]^(+).

Biological Testing Example 22 Evaluation of PPAR Activation In-Vitro

The assay was carried out in-vitro in three stable reporter cell lines,PPARα, PPARδ or PPARγ, expressing respectively a chimeric proteincontaining the ligand binding domain (LBD) of human PPARα, human PPARδor human PPARγ fused to the yeast transactivator GAL4 DNA binding domain(DBD).

The luciferase (Luc) reporter gene is driven by a pentamer of the GAL4recognition sequence in front of a β-globin promoter. The use ofGAL4-PPARα, GAL4-PPARδ and GAL4-PPARγ chimeric receptors allows forelimination of background activity from endogenous receptors andquantitation of relative activity across the three PPAR subtypes withthe same reporter gene.

Two unsubstituted reference substances, Reference 1 and 2, and five testsubstances, (7), (10), (11), (24) and (25) were tested in aconcentration of 10 μM. The structural formulae of the tested substancesare as show below:

The PPAR selectivity of the substances was determined by comparison toknown drug references (1 μM GW7647 for PPARα, 1 μM L-165041 for PPARδand 1 μM BRL49653 for PPARγ) set of 100% activity.

The results are presented in FIG. 1.

Example 23 Evaluation of PPARα Activation In-Vitro (ConcentrationResponse Data)

The assay was carried out in-vitro using mammalian-one-hybrid assays(M1H) comprising GAL4-DNA binding domain-PPARα-LBD fusion constructs inconjunction with 5×GAL4-sites driven Photinus pyralls luciferasereporter construct in transiently transfected HEK293 cells.

Compound (12) and positive control (GW7647) were tested at differentconcentrations. The results are presented in Table 1.

PPARα Compound EC50 (nM) Efficacy (%) GW7647 0.45 100 (12) 286 84

Example 24 Evaluation of the Effects on Lipid Metabolism In-Vivo in aDyslipidemic Model (APOE*3Leiden Transgenic Mice)

This animal model has proven to be representative for the humansituation regarding plasma lipoprotein levels, lipoprotein profiles, itsresponsiveness to hypolipidemic drugs (like statins, fibrates etc.) andnutrition. In addition, depending on the level of plasma cholesterolAPOE*3Leiden mice develop atherosclerotic lesions in the aortaresembling those found in humans with respect to cellular compositionand morphological and immunohistochemical characteristics.

Female APOE*3Leiden mice were put on a semi-synthetic Western-type diet(WTD, 15% cocoa butter, 40% sucrose and 0.25% cholesterol; all w/w).With this diet the plasma cholesterol level reached mildly elevatedlevels of about 12-15 mmol/l. After a 4 weeks run-in period the micewere sub-divided into groups of 10 mice each, matched for plasmacholesterol, triglycerides and body weight (t=0).

The test substances were administered orally as admix to theWestern-type diet. To facilitate the mixing of the compounds sunfloweroil was added to a total oil volume of 10 mL/kg diet.

After three weeks of treatment (t=3 weeks) mice were fasted overnight(o/n) and blood samples were taken to measure plasma ketone bodies andfree fatty acids. At t=0 and 4 weeks blood samples were taken after a 4hour-fast period to measure plasma cholesterol and triglycerides.

Two unsubstituted reference substances, Reference 3 and 2, and threetest substances, (7), (10) and (12), were dosed at 0.3 mmol/kg bw/day.The structural formulae of the tested substances are as show below:

The results are shown in FIG. 2.

Example 25 Evaluation of the Effects on Glucose Metabolism in a DiabetesType-II Model (Male Ob/Ob Mice)

Ob/ob mice can be used as a model for type II diabetes. The mice arehomozygous for the obese spontaneous mutation (Lepo) leading to leptindeficiency. In addition to obesity (ob/ob mice may reach three times thenormal body weight of wild type controls), ob/ob mice exhibit a diabetestype II-like syndrome of hyperglycemia, glucose intolerance, elevatedplasma insulin, infertility, impaired wound healing, and an increase inhormone production from both pituitary and adrenal glands.

Male ob/ob mice were put on a normal low-fat diet for a few weeks foracclimatization. After the acclimatization period the mice weresub-divided into three groups of 10 mice each, matched for body weight,plasma glucose and insulin (t=0).

All compounds were administered orally as admix to AM II diet. Tofacilitate the mixing of the compounds, sunflower oil was added to atotal oil volume of 10 ml/kg diet.

At t=0, 2 and 4 weeks body weight and food intake was measured. At t=0,2 and 4 weeks blood samples were taken after a 4 hour-fast period tomeasure whole blood HbAlc and plasma glucose, insulin, cholesterol andtriglycerides.

Pioglitazone was used as reference (15 mg/kg bw/day). Compound (10) wasdosed at 0.6 mmol/kg bw/day. The results (t=4) are shown in FIGS. 3-6.

1-86. (canceled)
 87. A method for the treatment of a disease orcondition chosen from a dyslipidemic condition; elevated triglyceridelevels, LDL cholesterol levels, and/or VLDL cholesterol levels;peripheral insulin resistance; and/or a diabetic condition in a subjectin need thereof, comprising administering to the subject apharmaceutically active amount of

2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoic acid.88. The method according to claim 87, wherein the dyslipidemic conditionis hypertriglyceridemia (HTG).
 89. The method according to claim 87,where the diabetic condition is type 2 diabetes.
 90. A method for thereduction of at least one of plasma insulin, blood glucose, or serumtriglycerides in a subject in need thereof, comprising administering tothe subject a pharmaceutically active amount of

2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoic acid.91. A method for the treatment of atherosclerosis in a subject in needthereof, comprising administering to the subject a pharmaceuticallyeffective amount of a compound of formula (I):

wherein R₁ is chosen from a C₁₀-C₂₂ alkenyl group having 3-6 methyleneinterrupted double bonds in Z configuration and a C₁₀-C₂₂ alkynyl grouphaving 1-6 triple bonds; R₂ and R₃ are the same or different and eachare independently chosen from a hydrogen atom, a hydroxy group, an alkylgroup, a halogen atom, an alkoxy group, an acyloxy group, an acyl group,an alkenyl group, an alkynyl group, an aryl group, an alkylthio group,an alkoxycarbonyl group, a carboxy group, an alkylsulfinyl group, analkylsulfonyl group, an amino group, or an alkylamino group, providedthat R₂ and R₃ are not both a hydrogen atom; or R₂ and R₃ are connectedin order to form a cycloalkane; Y is chosen from sulphur, sulfoxide, andsulfone; and X is a carboxylic acid or a derivative thereof, wherein thederivative is a carboxylic ester, a carboxamide, a monoglyceride, adiglyceride, a triglyceride, or a phospholipid; or a pharmaceuticallyacceptable salt thereof.
 92. The method of claim 91, wherein thecompound of formula (I) is

2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoic acid.93. A method for the treatment myocardial infarction and/or aninflammatory disease or condition in a subject in need thereof,comprising administering to the subject a pharmaceutically effectiveamount of a compound of formula (I):

wherein R₁ is chosen from a C₁₀-C₂₂ alkenyl group having 3-6 methyleneinterrupted double bonds in Z configuration and a C₁₀-C₂₂ alkynyl grouphaving 1-6 triple bonds; R₂ and R₃ are the same or different and eachare independently chosen from a hydrogen atom, a hydroxy group, an alkylgroup, a halogen atom, an alkoxy group, an acyloxy group, an acyl group,an alkenyl group, an alkynyl group, an aryl group, an alkylthio group,an alkoxycarbonyl group, a carboxy group, an alkylsulfinyl group, analkylsulfonyl group, an amino group, or an alkylamino group, providedthat R₂ and R₃ are not both a hydrogen atom; or R₂ and R₃ are connectedin order to form a cycloalkane; Y is chosen from sulphur, sulfoxide, andsulfone; and X is a carboxylic acid or a derivative thereof, wherein thederivative is a carboxylic ester, a carboxamide, a monoglyceride, adiglyceride, a triglyceride, or a phospholipid; or a pharmaceuticallyacceptable salt thereof.
 94. The method according to claim 93, for thetreatment of myocardial infarction.
 95. The method according to claim93, for the treatment of an inflammatory disease or condition.
 96. Themethod of claim 94, wherein the compound of formula (I) is

2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoic acid.97. The method of claim 95, wherein the compound of formula (I) is

2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoic acid.98. A pharmaceutical composition comprising the lipid compound

2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenylthio)butanoic acid.